Next-generation sequencing identifies the natural killer cell microRNA transcriptome

Natural killer (NK) cells are innate lymphocytes important for early host defense against infectious pathogens and surveillance against malignant transformation. Resting murine NK cells regulate the translation of effector molecule mRNAs (e.g., granzyme B, GzmB) through unclear molecular mechanisms. MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate the translation of their mRNA targets, and are therefore candidates for mediating this control process. While the expression and importance of miRNAs in T and B lymphocytes have been established, little is known about miRNAs in NK cells. Here, we used two next-generation sequencing (NGS) platforms to define the miRNA transcriptomes of resting and cytokine-activated primary murine NK cells, with confirmation by quantitative real-time PCR (qRT-PCR) and microarrays. We delineate a bioinformatics analysis pipeline that identified 302 known and 21 novel mature miRNAs from sequences obtained from NK cell small RNA libraries. These miRNAs are expressed over a broad range and exhibit isomiR complexity, and a subset is differentially expressed following cytokine activation. Using these miRNA NGS data, miR-223 was identified as a mature miRNA present in resting NK cells with decreased expression following cytokine activation. Furthermore, we demonstrate that miR-223 specifically targets the 3′ untranslated region of murine GzmB in vitro, indicating that this miRNA may contribute to control of GzmB translation in resting NK cells. Thus, the sequenced NK cell miRNA transcriptome provides a valuable framework for further elucidation of miRNA expression and function in NK cell biology

Homozygous KSR1 deletion attenuates morbidity but does not prevent tumor development in a mouse model of RAS-driven pancreatic cancer.

Given the frequency with which MAP kinase signaling is dysregulated in cancer, much effort has been focused on inhibiting RAS signaling for therapeutic benefit. KSR1, a pseudokinase that interacts with RAF, is a potential target; it was originally cloned in screens for suppressors of constitutively active RAS, and its deletion prevents RAS-mediated transformation of mouse embryonic fibroblasts. In this work, we used a genetically engineered mouse model of pancreatic cancer to assess whether KSR1 deletion would influence tumor development in the setting of oncogenic RAS. We found that Ksr1-/- mice on this background had a modest but significant improvement in all-cause morbidity compared to Ksr1+/+ and Ksr1+/- cohorts. Ksr1-/- mice, however, still developed tumors, and precursor pancreatic intraepithelial neoplastic (PanIN) lesions were detected within a similar timeframe compared to Ksr1+/+ mice. No significant differences in pERK expression or in proliferation were noted. RNA sequencing also did not reveal any unique genetic signature in Ksr1-/- tumors. Further studies will be needed to determine whether and in what settings KSR inhibition may be clinically useful

Allosteric Activation of Functionally Asymmetric RAF Kinase Dimers

Although RAF kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood. Recently, dimerization was shown to be important for activation. Here we show that the dimer is functionally asymmetric with one kinase functioning as an activator to stimulate activity of the partner, receiver kinase. The activator kinase did not require kinase activity but did require N-terminal phosphorylation that functioned allosterically to induce cis-autophosphorylation of the receiver kinase. Based on modeling of the hydrophobic spine assembly, we also engineered a constitutively active mutant that was independent of Ras, dimerization, and activation-loop phosphorylation. As N-terminal phosphorylation of BRAF is constitutive, BRAF initially functions to activate CRAF. N-terminal phosphorylation of CRAF was dependent on MEK, suggesting a feedback mechanism and explaining a key difference between BRAF and CRAF. Our work illuminates distinct steps in RAF activation that function to assemble the active conformation of the RAF kinase

Availability of Advanced Breast Imaging at Screening Facilities Serving Vulnerable Populations

ObjectiveAmong vulnerable women, unequal access to advanced breast imaging modalities beyond screening mammography may lead to delays in cancer diagnosis and unfavourable outcomes. We aimed to compare on-site availability of advanced breast imaging services (ultrasound, magnetic resonance imaging [MRI], and image-guided biopsy) between imaging facilities serving vulnerable patient populations and those serving non-vulnerable populations.Setting73 imaging facilities across five Breast Cancer Surveillance Consortium regional registries in the United States during 2011 and 2012.MethodsWe examined facility and patient characteristics across a large, national sample of imaging facilities and patients served. We characterized facilities as serving vulnerable populations based on the proportion of mammograms performed on women with lower educational attainment, lower median income, racial/ethnic minority status, and rural residence.We performed multivariable logistic regression to determine relative risks of on-site availability of advanced imaging at facilities serving vulnerable women versus facilities serving non-vulnerable women.ResultsFacilities serving vulnerable populations were as likely (Relative risk [RR] for MRI = 0.71, 95% Confidence Interval [CI] 0.42, 1.19; RR for MRI-guided biopsy = 1.07 [0.61, 1.90]; RR for stereotactic biopsy = 1.18 [0.75, 1.85]) or more likely (RR for ultrasound = 1.38 [95% CI 1.09, 1.74]; RR for ultrasound-guided biopsy = 1.67 [1.30, 2.14]) to offer advanced breast imaging services as those serving non-vulnerable populations.ConclusionsAdvanced breast imaging services are physically available on-site for vulnerable women in the United States, but it is unknown whether factors such as insurance coverage or out-of-pocket costs might limit their use

<i>Pdx1-Cre;LSL-Kras</i>^{<i>G12D/+</i>}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>-/-</i> mice develop pancreatic tumors.

<p>H&E (top), pERK (middle) and Ki67 (bottom) staining of tumors harvested from <i>Pdx1-Cre</i>;<i>LSL</i>-<i>Kras</i>^{<i>G12D/</i>+}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>+/+</i>, <i>Ksr1</i>^<i>+/-</i>, and <i>Ksr1</i>^<i>-/-</i> mice (bar = 100μm). Panels to the right show magnification of indicated region (bar = 40μm). Tumors have moderately differentiated ductal morphology that stains strongly for pERK, accompanied by stromal desmoplasia. Ki67 stains some ductal and surrounding cells.</p

<i>Pdx1-Cre;LSL-Kras</i>^{<i>G12D/+</i>}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>-/-</i> mice develop PanIN lesions at similar rates and severity to <i>Ksr1</i>^<i>+/-</i> and <i>Ksr1</i>^<i>+/+</i> mice.

<p>H&E staining of pancreatic tissues from <i>Pdx1-Cre</i>;<i>LSL</i>-<i>Kras</i>^{<i>G12D/</i>+}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>+/+</i>, <i>Ksr1</i>^{<i>+/ -</i>}, and <i>Ksr1</i>^<i>-/-</i> mice sacrificed at 3 months of age highlights PanIN lesions surrounded by normal tissue (bar = 40μm).</p

<i>Pdx1-Cre</i>;<i>LSL</i>-<i>Kras</i>^{<i>G12D/</i>+}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>-/-</i> mice have a modest but statistically significant decrease in all-cause morbidity.

<p>A. Kaplan-Meier curves for <i>Pdx1-Cre</i>;<i>LSL</i>-<i>Kras</i>^{<i>G12D/</i>+}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>+/+</i> and <i>Ksr1</i>^<i>+/-</i> mice based on age at sacrifice or death. 8 <i>Ksr1</i>^<i>+/-</i> and 10 <i>Ksr1</i>^<i>+/+</i> mice had to be censored. Median age at sacrifice or death was 152 days for <i>Ksr1</i>^<i>+/-</i> mice and 160 days for <i>Ksr1</i>^<i>+/+</i>mice; there was no statistically significant difference between the two groups (p = 0.4683 by log-rank test). B. Kaplan-Meier curves for <i>Pdx1-Cre</i>;<i>LSL</i>-<i>Kras</i>^{<i>G12D/</i>+}<i>;Trp53</i>^{<i>flox/wt</i>}<i>;Ksr1</i>^<i>-/-</i>, and <i>Ksr1</i>^<i>+/-</i> combined with <i>Ksr1</i>^<i>+/+</i> mice based on age at sacrifice or death. 3 <i>Ksr1</i>^<i>-/-</i> were censored. There was a modest but statistically significant difference between median age at sacrifice or death for <i>Ksr1</i>^<i>-/-</i> mice and the control group (191 and 159 days, p = 0.0344 by log-rank test).</p

Allosteric Activation of Functionally Asymmetric RAF Kinase Dimers

Although RAF kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood. Recently, dimerization was shown to be important for activation. Here we show that the dimer is functionally asymmetric with one kinase functioning as an activator to stimulate activity of the partner, receiver kinase. The activator kinase did not require kinase activity, but did require N-terminal phosphorylation that functioned allosterically to induce cis-autophosphorylation of the receiver kinase. Based on modeling of the hydrophobic spine assembly, we also engineered a constitutively active mutant that was independent of Ras, dimerization, and activation loop phosphorylation. Since N-terminal phosphorylation of BRAF is constitutive, BRAF initially functions to activate CRAF. N-terminal phosphorylation of CRAF was dependent on MEK suggesting a feedback mechanism and explaining a key difference between BRAF and CRAF. Our work illuminates distinct steps in RAF activation that function to assemble the active conformation of the RAF kinase